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Full text of "Etiology of false broomrape of tobacco"

The Etiology of False Broomrape 
of Tobacco 



By 
WAYNE C. MIXSON 



A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF 
THE U-NIVERSITY OF FLORIDA IN PARTIAL 
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF 
DOCTOR OF PHILOSO.^HY 



UNIVERSITY OF FLORIDA 
1972 



ACKNOWLEDGMSXTS 

The author wishes to express his thanks to Dr. 
Carol R, Killer, former Supervistory Couumittee Chairman 
and assistant professor of plant pathology at the 
University of Florida, for his assistance during this 
study. Special thanks go to Dr. R. E. Stall, dairnian 
of the Supervisory Committee, for his guidance and 
advice as well as critical assistance during the prep- 
aration of this manuscript. Thanks also go to the 
other members of the S'upervisory Committee, Dr. V, G. 
Perry, Dr. L. H. Purdy, and Fred Clark, 

Acknowledgments are also extended to Forrest 
Parrott of Tabacalera Nicaraguence, Managua, Nicaragua, 
and Paul Gardner, Tabacalera Hondurena, San Pedro Sula, 
Honduras, for their assistance during the author's 
trips to Central America. Special consideration is 
also extended to the British American Tobacco Company 
for making the author's trips to Central America possible. 



11 



TABLE OF CONTENTS 

Page 
ACKNOWLEDGMENTS j_i 

LIST 0? FIGURES ^ 

LIST OF TABLES ^^^ 

ABSTRACT ^^^^ 

INTRODUCTION 

MATERIALS AND METHODS 5 

Inoculation and Transmission 

Methods 5 

Sources and Types of FBR Tissue 6 

Effect of Temperature and Size 

of Plant on Fasciation 12 

Host Range and Resistance 

Within Nicotiana species and 

Cultivars of N. tabacum 17 

Effect of Fertilization Levels 

on FBR Development 17 

Determination and Isolation of 

Causal Agent j^g 

RESULTS 

21 

Inoculation and Transmission 

Methods „- 

Sources and Types of FBR Tissue 23 

Effect of Temperature, Time 
From Inoculation, and Size 
of Plants on Fasciation 27 



111 



Page 

Host Range and Resistance 

Within Nicotiana Species and 

Cultivarc of N. tabacurn 30 

Effect of Fertilization Levels 

on FBR Development ....,,..., 32 

Isolation of the Causal Agent 32 

Identification of FBR-72-65. ....... 38 

DISCUSSION 40 

SUlvfl-IARY 42 

LITERATURE CITED 44 

BIOGRAPHICAL SKETCH 47 



IV 



Figure 



10 



LIST OF FIGURES 



Burley tobacco plants in a 
Kentucky field stunted 
by FBR 



Fasciated tissue from burley 
tobacco plants in Kentucky 
infected with FBR 



Fasciated tissue from flue- 
cured in Honduras infected 
with FBR 



Hardened FBR galls on roots 
of tobacco 



Page 



Fasciated tissue from burley 
tobacco plants in Nicaragua 
infected with FBR q 

Fasciated tissue from cigar 
wrapper tobacco in Nicaragua 
infected with FBR 9 

Above ground plants 

originating from below 

ground fasciated tissue 

caused by FBR , -^q 



11 



Cauliflower plant found 
naturally infected with 
FBR in Nicaragua 23 



14 



Succulent FBR galls on 

roots of tobacco ^5 

Succulent leafy FBR tissue 
and small plants originating 
from underground tissue on 
roots of tobacco ^g 



Figure ■ page 

11 Severely FBR infected root 

systems resulting in 

stunting of burley tobacco 

cor.rj-.red to uninfected 

plant in Nicaragua ....... 24 

12 Small plants forming from 

fasciated tissue near the 

soil surface on burley 

tobacco in Nicaragua 25 

13 Root systems of FBR inoculated 

tobacco plants grown at 25 C 

(right), 30 C (center), 

and 35 C (left) 29 

14 FBR symptoms produced by 

inoculation of tobacco 

roots with bacterial 

isolate, FBR-70-33 35 

15 FBR symptoms produced by 

inoculation of tobacco 

roots with bacterial 

isolate, FBR-72-65 36 



VI 



Table 



LIST OF TABLES 



Page 

Effect of dilution of crude 
extract of false broomrape 
tissue on percent infection 
of tobacco roots ...,.,,, 22 

Reaction of the tobacco 
cultivar Hicks to inoc- 
ulation with false broomrape 
at three temperature regimes 
and three time periods 28 

Reaction of several genera of 
plants including Nicotiana 
species and N, tabacum 
cultivars. ,~ 31 

Effect of fertilization levels 
on false broomrape develop- 
ment of burley and flue-cured 
tobacco. ..... 33 

Results of physiological. Gram 
stain, and optimum temper- 
ature tests conducted on 
known C. fascians and bacterial 
isolate, FBR- 72-65 39 



VI 1 



Abstract of Dissertation Presented to the Graduate Council 
of the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 

THE ETIOLOGY OF FALSE BROOMRAPE 
OF TOBACCO 

By 

Wayne C. Mixson 

December, 1972 

Chairraanj Dr. R. E. Stall 

Major Departments Plant Pathology 

The false broomrape (FBR) pathogen was easily 
transmitted from tobacco to tobacco using any type of 
fasciated tissue other than the small plants formed 
above ground from the fasciated tissue near the soil 
surface. The best method for inoculation was to 1) grind 
FBR tissue in a sterile mortar, with additions of small 
amounts of 0.1 M potassium phosphate buffer at pH 7.0 
until the horaogenate has a consistency of a thick paste; 
2) carelessly remove 20 cm tall plants from soil; 3) dust 
clean roots with 600 mesh carborundum and rub the roots 
gently with a cotton swab; 4) immerse the roots of the 
plants immediately in the fresh FBR homogenate and rub 
the roots gently with a cotton swab; and 5) transplant 
the inoculated plants immediately. This method gave 100% 
transmission. No differences were found between FBR 
from the United States and Central America. FBR development 



Vlll 



is associated with the temperature most favorable for 
plant growth. At least 4 weeks were required for gall 
development. All species of Nicotiana tested were 
susceptible to FBR as were all 14 cultivars of N. 
tabacum tested. Cauliflower with FBR was observed 
growing in Nicaragua. Sweet potato, cabbage, and sweet 
pea were susceptible to FBR in greenhouse inoculations. 
A bacterium was isolated from FBR tissue and upon sub- 
sequent inoculations caused FBR symptoms on tobacco. 
This isolate, FBR-72-65, was identified as a species 
of Corynebacterium and is probably C. fascians . A 
known isolate of C. fascians (ICPP CF-19) caused similar 
symptoms on tobacco. Two colony types were observed 
(smooth and rough) for FBR-72-65 and ICPP CF-19 and 
only the smooth types were pathogenic. Results of 
all physiological tests with the FBR bacterium agreed 
with the Sergey's Manual description of C. fascians 
except for nitrate reduction. The known C. fascians 
isolates also were anomolous with the nitrate reduction 
test. The best medium for isolation of the causal 
bacterium was found to be one containing 40 g Difco PDA, 
2 g Na2HP0^, 2 g NaCl, 1 g sodium citrate, 1 g asparagine, 
and 300 mg potassium dichromate per 1000 ml of water. 



IX 



INTRODUCTION 

False broomrape (FBR) is a disease of the root 
system of tobacco (Nicotiana tabacum L.)- Symptoms 
of FBR include a fasciation of the root tissue in 
which masses of white, succulent, and irregular out- 
growths form. Under certain conditions differentiation 
of chlorophyll containing leaves (15) and/or a complete 
plant may occur from the outgrowths. 

Thompson (19) placed FBR tumors into the following 
three groups: 1) heterotopic teratomas, 2) leafy 
teratomas, and 3) secondary root teratomas. All the 
galls, except those on the main root (heterotopic 
teratomas), had tumor gaps and traces connecting the 
tumors to the vascular system of the tobacco plant. 

Valleau (22) named the disease false broomrape 
because he first identified the disease as broomrape 
which is caused by Orobanchi spp . , a parasitic seed 
plant. Thompson (19) stated that the tumors are some- 
what similar to crown gall caused by Aqrobacterium 
tumefaciens (Smith and Town) Conn and tumors that are 
hereditary in nature. FBR appears to have different 
etiology from either of these two tumor problems, however. 

FBR was first reported in Kentucky on burley tobacco 
in 1951 (22) and hc.^ since been reported in Florida (24), 



North Carolina (8), Georgia (6), and Maryland (18). It 
has caused little economic loss to the total tocacco 
crop in the United States (12), however. Jenkins (6) 
reported that the disease was sporadic and never caused 
any serious losses in Georgia and Massey (U) found 
the same to be true in Kentucky. 

Economic losses do occur in individual fields, 
however. Plants in some fields in Kentucky were ob- 
served in 1969 by the author to be severely diseased 
(Fig. 1). It was estimated tnat the yield in these 
fields would be reduced at least 20%. Tne Diseased 
plants were 1 to 3 ft in height while the healthy plants 
were 5 to 6 ft in height. When the root systems on the 
stunted plants were examined, all of them had galls 
ranging in diameter from 2 to 8 cm on the tap root. 

The most severe losses apparently occur in Central 
America where FBR has been observed in Honduras, Nicaragua, 
El Salvador, and Panama (12, 15). In the Central American 
countries FBR is more severe on burley than flue-cured 
tobacco. The author also observed FBR on shade tobacco 
in the burley area of Nicaragua in 1970 and 1971, A 
severe outbreak in the Jalapa Valley resulted in a 28% 
reduction in yield (15). 

Several researchers have tried and failed to isolate 
a causal agent of FBR (3, 8, 19). Some proposed that FBR 
is caused by the bacterium, C. fascians Tilford (Dowson) 
(8, 12, 17, 19, 22). Valleau noted the similarity of 
this disease to leafy gall of tobacco described by Tilford (20) 




Figure 1 . Burley tobacco plants in a 
Kentucky field stunted by 
FBR. 



.-=^/./i 



and Lacey (iO). Thompson (19) and Newman (17) inoc- 
ulated tobacco with C. fascians and obtained symptoms 
almost identical to FBR but were unable to complete 
Koch's postulates. Kelman (8) did the same but did not 
believe that C. fascians was the causal organism because 
of differences in symptoms and the fact that FBR developed 
only after wounding, whereas C, fascians did not. 

C. fascians was first identified as a pathogen of 
sweet pea by Tilford (20) and was found to cause root 
fasciations, leafy galls, and several other disorders 
on many plants particularly on ornamental plants (1, 16). 
Jacobs and Mohanty (5) found that C. fascians has a very 
wide host range >and confirmed the existence of host 
specificity among strains of the pathogen. Lacey (10) 
isolated a bacterium associated with leafy gall of sweet 
peas and found that these strains were pathogenic on 
Nicotiana qlutinosa and N. tabacum . vicia faba L. and 
Phaseolu s vulgaris L. were susceptible to the tobacco 
strain (10). 

FBR has the potential of becoming a serious disease 
on tobacco in the United States as well as on other 
economic crops. As a result of a review of the literature 
which revealed that seve-al researchers have conflicting 
views as to the causal agent of the disease and subsequent 
personal observations of the disease in fields of tobacco 
in Kentucky and Central America, the author conducted 
research to assist in identifying the causal agent 
which, in turn, might aid in its control. 



hiATERIALS AND METHODS 

Iioculation and Transmission Methods 

In initial experiments inoculation techniques for 
transmission of FBR to tobacco were with macerated, 
fasciated tissue because no causal agent had been iden- 
tified. Transmission of the causal agent of FBR by 
method described by Dukes was tried (3). The Dukes* 
method is 1) grind FBR tissue in a sterile mortar, with 
additions of small amounts of 0.1 M potassivun phosphate 
buffer at pH 7.0; 2) grind until the homogenate has the 
consistency of a thick paste j 3) dust clean roots of 
healthy tobacco plants in the 4-5 leaf stage of growth 
that were carefully removed from the soil with 600 mesh 
carborundum and rub the roots gently with cotton swabs; 
4) immerse the roots of the plants immediately in the 
fresh FBR homogenate and rub the roots gently with a 
cotton swab; and 5) transplant the inoculated plants 
iiTimediately. Kelman's method (8) of inoculation was also 
tried. This method was simpler in that the plants' roots 
were wounded and immersed in a suspension of fragments of 
FBR tissue. 

Several variations of Dukes' method were also 
tested. These were 1) identical procedure but the 
plants were pulled carelessly from the soil; 2) the 
roots were deliberately rubbed for injury; 3) the same 

5 



as number 1 without carborundum; 4, 5, 6) repeat the 
above three metnods but the homogenata had been filtered 
througn Whatman no. 1 filter paper; 7) identical to 
number 1 but with homogenate diluted lilO, lilO^, lilO^, 
lilO , and lilO^j 8) inject homogenate into roots with a 
hyperdermic syringe and 27 gauge needle; and 9) transplant 
in naturally and artifically infested soil (plants were 
pulled from soil, soil carefully washed from roots, and 
then rubbed with carborundtun) . Method 1 was used to 
inoculate plants in subsequent tests unless otherwise 
indicated, and the cultivar Kicks was also used unless 
otherwise indicated. 

Sources and Types of FBR Tissue 

Fasciated tissue was obtained from burley tobacco 
in Kentucky and Nicaragua (Fig. 2 and 3), cigar wrapper 
tobacco in Nicaragua (Fig. 4 and 5), and flue-cured 
tobacco in Honduras (Fig. 6). FBR was observed under 
natural conditions at these locations for differences in 
symptoms. Tissue was obtained from many fields in 
Nicaragua and Honduras and kept separate to screen for 
possible differences that might exist. Tobacco plants 
were inoculated with each of the above sources of 
diseased materials, grown in a greenhouse for several 
weeks, and observed for symptom expression. Tissue 
from the fasciated roots of a cauliflower ( Brassica 
plearacea var. botrytis L.) plant found in Nicaragua 




Figure 2. Fasciated tissue from bur ley 
tobacco plants in Kentucky 
mtected with FBR, 




Figure 3. Fasciated tissue from burley 
tobacco plants in Nicaragua 
infected with FBR. 




Figure 4. Fascia ted tissue from cigar 
wrapper tobacco in Nicaragua 
infected with FBR. 



10 




Figure 5, Above ground plants originating 
from below ground fasciated 
tissue caused by FBR. 



il 




Figure 6. Fasciated tissue from flue-cured 
tobacco in Honduras infected 
with FBR. 



12 

was also used to test for transmission to tobacco (Fig. 7), 

Tobacco plants were inoculated with various types of 
fasciated tobacco tissue. This tissue was either dried 
or living. The living tissue was further subdivided 
into hardened galls (Fig. 8), succulent galls (Fig, 9), 
succulent leafy tissue (Fig. lO), and small plants that 
had developed from galls when they occurred near the soil 
surface (Fig. 10). The galls were also sectioned to 
obtain inner and outer tissue. 



Effect of Temperature and Size 
of Plant on B'asciation 



Fifteen tobacco plants of the Hicks cultivar were 
inoculated and placed in each of three growth chambers 
set at 25, 30, and 35 C. Five plants were removed at 
each of three time intervals of 3, 4, and 6 weeks. The 
plants were weighed and examined for fasciation. Any 
galls were then removed and weighed. Inoculated plants 
were compared with uninoculated checks. This experiment 
was repeated. 

Tobacco plants of the same cultivar at heights of 
10, 20, 40, 60, 80, and 100 cm were inoculated to deter- 
mine the optimum size for FBR development. Inoculated 
plants were held in a greenhouse, and the root systems 
were observed at intervals of 3, 4, 5, and 6 weeks. 




Figure 7, Cauliflower plant found naturally- 
infected with FBR in Nicaragua. 




Figure 8. Hardened FBR galls on roots of 
tobacco. 



15 




Figure 9. Succulent FBR galls on roots 
of tobacco. 



16 




Figure 10, 



Succulent leafy FBR tissue and 
small plants originating from 
underground tissue on roots of 
tobacco. 



17 



Host Range and Resistance VJithin Nicotiana 
Species and Cultivars of N« tabacum 

Two species, N. glutinosa L. and N. rustica L. , 
were inoculated along with the following cultivars of 
N. tabacum i Coker 80-F, F-17, N. C. 95, Hicks, Va 115, 
N. C. 2326, Coker 254, Coker 319, Coker 298, Speight 
G-7, McNair 14, Maryland 64, Bur ley 21, and Pennbel 69. 
Peanut (Archis hypogea L.), bean ( Phaseolus vulgaris L.), 
cauliflower, sweet potato ( Ipomoea batatus L.), cabbage 
( Brassica olearacea var. capita ta L.), sweet pea ( Lathyrus 
odoratus L.), and garden pea ( Pi sum sativium L. ) were also 
inoculated. Plants of the above species were root inoc- 
ulated and the sweet and garden pea plants were also inoc- 
ulated as described by Jacobs and Mohanty (5)» In this 
method the seeds were soaked in a slurry of FBR tissue 
^ and planted in sterile soil in the greenhouse. 

Effect of Fertilization Levels 
on FBR Development 

Twenty- five tobacco plants approximately 20 cm in 
height were inoculated with a slurry of FBR tissue and 
planted in 4-inch clay po^s containing sterile soil in 
the greenhouse. Five different fertilization treatments 
were established within each group of 25 plants 
(5 plants/treatment). The treatments were no fertilizer, 
1.5, 3, 4.5, and 6 g/pot of 3-6-9 analysis. Plants were 
maintained for 5 weeks following inoculation and the plants' 
roots, shoots, and resulting galls were weighed. Twenty-five 



18 

uninoculated plants were also planted in the soil with 
the five fertilizer treatments, 

« 

Determination and Isolation of 
Causal Agent 

Since several researchers have speculated that FBR 
is caused by a bacterium (3, 4, 8, 12, 17, and 19), 
experiments were designed to eliminate other causal agents 
and to isolate a causal agent. A slurry of FBR tissue 
was made and filtered through a Whatman no. 1 filter 
paper several times to remove plant material. The re- 
sulting filtrate was then filtered through a 0.45/a 
Millipore filter to remove any bacteria that were present. 
Presumably a virus would pass through into the filtrate. 
Roots of tobacco plants were inoculated with 1) the slurry 
before the first filtration, 2) the filtrate obtained 
through the Whatman filter, and 3) the filtrate obtained 
through the Millipore filter. A suspension containing 
tobacco mosaic virus (TMV) was also passed through a 0.45^ 
Millipore filter and then inoculated on tobacco to deter- 
mine, if indeed, a virus particle did pass through the 
filter. As a check the FBR filtrate obtained after 
Millipore filtration was plated on nutrient agar to deter- 
mine if any bacteria were allowed through. 

Streptomycin was added to a FBR filtrate through 
Whatman no. 1 paper to obtain the following concentrations! 
ppm, 50 ppm, 100 ppm, 200 ppm, 300 ppm, and 400 ppm. 
Each of these was inoculated on roots of tobacco plants. 



19 

The plants were allowed to grow for 5 weeks and were 
observed for gall development. 

Attempts were made to isolate the causal organism, 
and most of these attempts were directed at the iso- 
lation of a bacterium. The procedure in the first attempt 
was similar to that used by Tilford (20) in 1936 in his 
original isolation of C. fascians from sweet peas. A 
large piece of tissue was taken from the base of the 
fasciated growth, dipped in alcohol, flamed, and then 
crushed in 5 ml of sterile water. After 2 to 4 hr one 
drop of the liquid was transferred with a sterile pipette 
to a sterile dish; two drops were placed in a second 
dish} and three drops were placed in a third. Potato 
dextrose agar (PDA) at 48 C was added to the plates 
and held at 25 C. 

This same procedure was used in other isolations 
except the drops were placed on solidified PDA and smeared 
with a sterile glass rod. This was also done without the 
2 to 4 hr waiting period and repeated on nutrient agar (NA). 

In another attempt the medium described by Burkholder 
(2) for the isolation of C. sepidonicum , the causal agent 
of tuber ring rot and wilt of potato, was used. To make 
Burkholder's medium approximately 300 g of sliced potatoes 
were boiled and the broth made up to a liter. To this was 
added 5 g peptone, 2 g Na2HP04, 2 g NaCl, 1 g sodium 
citrate, 1 g asparagine, 6 g dextrose, 12 g agar. 



20 

Burkholcer's medium was also tried using potassium 
cichrornate to retard the growth of saprophytes. This 
was used at 100 ypa, 2C0 ppm, 300 ppm, and 400 ppm, 

Kado a;id Hes>cetc*s laediLuu D2 (9;^ which was proposed 
for isolation of Corynebacteriurg spp . was tried. This 
mediun contained 10 g glucose, 4 g casein hydrolysate, 
2 g yeast extract, 1 g NH^^Cl, 0.3 g MgS0^.7H20, 5 g LiCl, 
1.2 g Tris, 40 mg polymyxin sulfate, 2 nig sodium azide, 
and 15 g agar por liter of water. This was adjusted to 
pH 7.8 with KCl before autoclaving. Polymyxin sulfate 
and sodium azide were added after the medium had cooled 
to 50 C. 

Each of the types of gall tissue listed previously 
was used in isolation attempts with each media. The most 
widely used method of isolation was to macerate a young, 
succulent gall in sterile water, filter through Whatman 
no. 1 filter paper, and then dilute to 1:10, lilO^, and 
1:10-'. Tnese dilutions were added to the plates with 
sterile pipettes in quantities of 0.1. 0.5, and 1 ml 
and smeared with a sterile glass rod. The plates were 
incubated at 25 or 30 C. 

At the beginning of the isolation experiments, dif- 
ferent bacterial colonies were removed from the plate, 
purified, and inoculated to tobacco. These were also 
stored in sterile water and held at 10 C. Eventually, 
colonies visible within 24 hr -.-re no longer isolated. 
Only the slower growing colonies were used, especially 
the ones that became visible in 3 or more days. 



RESULTS 
Inoculation and Transmission Methods 

Dukes* method of inoculation gave 100% transmission 
of FBR if the slurry was used within 1 hr after prep- 
aration. When the plants used in inoculation studies 
were carefully removed from the soil and not injured 
before inoculation, 80% of the inoculated plants had 
FBR, whereas 100% were diseased when the plants were care- 
lessly pulled from the soil or if they were injured by 
rubbing. This means that excessive injury to the plant 
was not required for transmission of FBR. Injury to 
the tap root produced a larger gall and more stunting 
than when the tap root was not injured. 

After filtration through Whatman no. 1 filter paper 

to remove coarse plant parts, the filtrate was diluted 

3 
it 10 before transmission dropped below 50%. The 1:1, 

IslO, and l:lo2 dilutions gave 100% transmission (Table 1). 

Inoculation of a specific root with FBR filtrate 

using a hypodermic needle was successful, and a dilution 

of 1:102 gave 100% transmission. . This is a very good 

method to use when a small amount of inoculum, is available. 

It can also be used without disturbing the plant because 

soil can be carefully removed from only a small portion 

21 



22 



Table 1 . Effect of dilution of crude extract 
of false broonirape tissue on percent infection of 
tobacco roots. 



Dilution 


% infection 


ItO 


100 


111 


100 


lilO 


.100 


ltl02 


100 


lil03 


48 


1«104 


15 


1»105 






23 

of the root system before inoculation. 

FBR was easily transmitted using either naturally 
or artifically infested soil. When plants were carefully 
removed from the soil and transplanted in naturally or 
artifically infested soil, the transmission was 65%. 
When the plants were carelessly pulled from the soil 
and then planted in infested soil, the transmission 
percentage rose to 100%. This methoc produced galls 
on the fine lateral roots more often than did dipping 
the root system in the FBR homogenate. 

Based on the above experiments the most efficient 
inoculation method was pulling the plants carelessly 
from the soil, washing the soil away, and then dipping 
the root system in a Is 10 dilution (filtered or unfiltered) 
of FBR homogenate. This method consistently gave 100% 
transmission. 

Sources and Types of FBR Tissue 

Field observations of FBR on burley and flue-cured 
tobacco disclosed no differences in symptoms. No 
differences were noted in symptoms in the United States 
or in Central America. The disease was more severe in 
Central America (Fig. 11) and has occurred every year 
for the past 10 years, whereas in the United States 
the disease may not appear every year. Also FBR affected 
plants tended to form small plants above the ground (Fig. 
12) in Central America more often than in the United States. 



, -'I •,. •' . 



24 




Figure 11. Severely FBR infected root 

systems resulting in stunting 
of burley tobacco compared 
to uninfected plant in 
Nicaragua. 



25 




Figure 12. 



Small plants forming from 
fasciated tissue near the 
soil surface on burley 
tobacco in Nicaragua. 



26 

The soils of the two areas were very similar. The main 
difference seemed to be the lack of cold in Central 
America, although other reasons may exist for more 
severe disease in Central America. 

SeverEil fields were observed in Honduras and 
Nicaragua, both with and without FBR. Analysis of soil 
from these fields was made before the crop was trans- 
planted. There was nothing significant about fields 
with or without FBR as far as elements, organic matter, 
or pH. 

Diseased roots were brought to the University of 
Florida from Kentucky and Central America. Tobacco 
plants inoculated with FBR homogenate from various 
fields and maintained in the greenhouse were no dif- 
ferent in symptomotology. Each source of fasciated 
tissue produced small tobacco plants from the fasciated 
tissue that occurs near the soil surface. 

The cauliflower plants found in Nicaragua with 
FBR symptoms evidently were affected by the FBR causal 
agent. It was readily transmitted to tobacco and back 
to cauliflower. The symptoms on tobacco inoculated with 
the cauliflower homogenate were very similar to the 
symptoms produced with FBR tissue from tobacco. 

FBR was successfully transmitted from all types 
of fasciated tissues but not from the shoots that 
developed from such tissues. Old galls were dried at 
room temperature and stored at room temperature for 18 



27 

months, and 50% transmissibility was still attained. FBR 
could be transmitted, with equal frequency, from hardened 
galls, succulent galls, and leafy underground tissue. 



Effect of Temperature, Time From Inoculation, 
and Size of Plants on Fasciation 



The growth of fasciated tissue was correlated with 
optimum conditions for growth of the tobacco plant. 
The results of the growth chamber experiments are given 
in Table 2 and Fig. 13. The optimum temperature for 
gall development was 25-30 C. The growth of the plants 
after inoculation was correlated with temperature more 
than with FBR, although the plants with the larger galls 
were slightly smaller at a given temperature than were 
uninoculated plants at the same temperature. 

No galls were observed to develop at any of the 
three temperatures during the first 3 weeKs following 
inoculation. At the end of 4 weeks galls were foiind 
at ju C and 35 C but not at 25 C. Plants at all three 
temperatures had FBR at the end of 6 weeks with plants 
at 35 C having a smaller number ana weight. 

Tobacco plants 20 cm in height proved optimal for 
inoculation purposes. Galls developed faster on plants 
of this height than on plants of any of the other 
heights. The larger plants, 40-60 cm tall, were better 
for point inoculation, but for screening purposes the 
20 cm plants were superior. The plants required at least 
4 weeks for symptom expression regardless of size. 



28 



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Figurs 13. 



Rcot systems of FER inoculated 
tobacco plants grovn at 25 C 
(right), 30 C (center), and 
35 C (left). 



30 

Host Range and Resistance V/ithin Nicotiana 
STDecies and Cultivars of N. tabacum 

Three species of Nicotiana were inoculated along 
with several other cultivars of N. tabacum and other 
genera of plants. These are listed in Table 3 along 
with their reaction to FBR, 

All three species of Nicotiana were equally sus- 
ceptible to inoculation with FBR tissue. Of the 20 
plants of each of the different cultivars of N. tabacum 
inoculated with FBR and grown in the greenhouse, five 
were removed from the soil, observed for FBR development, 
weighed, and measured at four different time intervals. 
No differences were noted in susceptibility among the 
14 cultivars (Table 3). All plants developed fasciated 
tissue at the same rate and none were more retarded in 
overall growth than the others. 

Three of the other genera of plants evaluated proved 
susceptible to FBR (Table 3). Cauliflower, which was 
observed naturally diseased with FBR in Nicaragua, was 
susceptible in greenhouse inoculations along with cabbage. 
After several attempts FBR was transmitted to sweet potato. 
Sweet pea plants appeared to be susceptible, although the 
fascia tion did not develop to the extent reported by Tilford 
(20), Mohanty (16), Jacobs (5), and Lacey (11), 
Baker (1) reported Phaseolus vulgaris to be susceptible 
to C, fascians, but the cultivar of bean used in this 
work was not susceptible to FBR transmission from tobacco. 
Neither garden pea nor peanut was diseased. 



31 



Table 3. Reaction of several genera of plants 
including Nicotiana species and N, tabacum c ultivars. 







Genera 








Reaction* 


Cauliflower 


Brassica olearacea 


var. 


botr 


ytis 


+ 


Peanut 


Arachis hypoqea 




Bean 


Phaseolus vulgaris 










Sweet potato 


xpomoea batatus 


var. 

m 


capi 


tata 


+ 


Cabbage 


Brassica olearacea 


+ 


Sweet pea 


Lathyrus odoratus 


^ 


Garden pea 


Pisurn sativium 


mtum 








Tomato 


Lycopersicon escute 


^^ 


Tobacco 


N. rustica 




•f 




N. CTlutinosa 








+ 




N. tabacuiTi 






Coker 80-F 








+ 




F-17 








+ 




N. C. 95 








+ 




Hicks 








+ 




Va 115 








+ 




N. C. 2326 








+ 




Coker 254 








^ 




Coker 319 








-i- 




Coker 298 








+ 




Speight G-7 








+ 




.. McNair 14 








4- 




Maryland 64 








+ 




Bur ley 21 








+ 


* + i nrsi na^J-oc 


Pennbel 69 








+ 



32 



Effoct of Fertilization Levels on 
FBR Develop;;-,ant 

Fertilization had no effect on the initiation of 
fasciation (Table 4). Increasing the fertilization 
rate of the tobacco plants did not result in FBR 
being initiated sooner. Higher rates of fertilization 
did result in a faster development of the fasciated 
tissue once it was initiated. 

Isolation of the Causal Agent 

FBR was rot transmitted to tobacco by using 
bacterial free filtrates. Neither was it transmitted 
using a Whatman filtrate treated with streptomycin at 
100 ppm and above. When the filtrate and treated fil- 
trate were plated on nutrient agar, few bacterial colonies 
grew and at 300 ppm no bacterial colonies grew. TMV 
particles passed through the Millipore filter since 
plants inoculated with TMV filtrate developed TMV, 
These two experiments indicate that a bacterium but not 
a virus is the causal agent. Ease of transmission would 
tend to rule out a mycoplasma as the causal agent. 

The mediura chosen for most of the isolation attempts 
contained the following per 1000 ml: 40 g Difco PDA, 
2 g Na2KP04, 2 g NaGl, 1 g sodium citrate, 1 g asparagine, 
and 100 mg potassium dichromate. Kado's medium, PDA, 
and NA were not successful because many saprophytic 
bacteria grew on them. 



33 



Table 4. Effect of fertilization levels on 
false broomrape development of burley an4 flue-cured 
tobacco. 





Fertility level* 
g/pot 


Tumor 


Weight (g) 




Burley 


Flue-cured 





5 


5 


1.5 


6 


7 


3.0 


10 


11 


4.5 


11 


13 


6.0 


13 


16 



34 

Seventy- three bacterial isolations were made from 
the different types of FBR tissue. Most were slow 
growing and creiae, beige, yellow, or ye How- orange in 
color. These isolates .ere stored in sterile water at 
10 C until the inoculated plants reacted postively or 
negatively after a 6 weoc period. 

The 33rd isolate obtained (FBR-70-33) was from a 
gall on a plant in the greenhouse induced by tissue from 
burley tobacco in Kentucky. After 4 weeks four out of 
five tobapcb plants inoculated with this isolate expressed 
symptoms of FBR (Fig. 14). Five more tobacco plants 
were then inoculated with FBR-70-33. No FBR symptoms 
were expressed after 7 weeks. This was repeated with the 
same results. Attempts to isolate the bacterium from the 
galls of FBR-70-33 inoculated plants were not successful. 

Isolation attempts were continued, and isolate 
FBR-72-65 was obtained from a hardened gall of burley 
tobacco brought from Nicaragua. This isolate appeared 
on the potassium dichromate medivun after 3 days as a 
pinpoint yellow-orange colony. This was the only colony 
of this type observed on any of the 20 plates that were 
smeared with 0.5 ml of the 1:10 dilution of the FBR 
homogenate previously described. This isolate stained 
Gram positive. All five plants inoculated with the 
isolate expressed FBR symptoms (Fig. 15). 

After a suspect bacterium was isolated, a culture of 
C. fascians was requested from M. P. Starr, Curator of the 



35 




Figure 14. 



FBR symptoms produced by 
inoculation of tobacco 
roots with bacterial 
isolate, FBR-70-33. 



36 




Figure 15. FBR symptoms produced by 
inoculation of tobacco 
roots with bacterial 
isolate, FBR-72.65. 



37 

International Collection of Plant Pathogenic Bacteria, 
Davis, California, for comparison. This isolate of C. 
fascians (ICPP CF-19) also produced FBR symptoms on 
tobacco plants. When the two bacteria, ICPP CF-19 and 
FBR-72-65, vGre cultured on Difco PDA, both turned 
deep orange after 5 days. When FBR-72-65 was observed 
with the aid of a dissecting microscope, two colony types 
were evident, one rough and one smooth. The majority 
of the colonies were of the rough type. When the C. 
fascians isolate obtained from Starr was observed under 
the microscope, the same two colony types were observed. 
But in this instance the majority were of the smooth 
type. The smooth isolate was then designated FBR-72-65-1S 
and the rough isolate, FBR-72-65-iR, 

Tobacco plants were inoculated with each colony type 
from both ICPP CF-19 and FBR-72-65. In both instances 
the smooth isolates proved pathogenic and the rough 
ones did not. Selective development of the rough isolate 
in culture probably explains why the original FBR-72-65 
and FBR-70-33 seemed to lose pathogenicity on subsequent 
inoculations. The smooth colony type was reisolated 
from galls caused by inoculation with FBR-72-65, and 
subsequent inoculations produced FBR symptoms. 

C. fascians grew on Burkholder 's medium with 
additions of up to JOO ppm potassium dichromate which 
severely retarded growth of saprophytic bacteria and 
fungi. With this medium reisolation of FBR-72-65 from 



38 



a gall resulting from inoculation by FBR-72-65 was 

accomplished . 

Identification of FBR-72-.65 

Physiological tests were undertaken using the known 
C. fascians (rough and smooth) and FBR-72-65-1S and -IR. 
These and the results are listed in Table 5 along with 
the cnaracterization from Bergey's Manual . All test 
results of the FBR-72-65-iS and -iR agreed with the 
results of the known C. fascians (rough and smooth). 
The nitrate reduction tests did not agree with Bergey's 
Manual . Based on these tests and comparisons with the 
known C. fascians, FBR-72-65 is a species of Corynebacterium 
and in all likelihood is C, fascians. 



39 



Table 5. Results of physiological. Gram stain, 
and optimum temperature tests conducted on icnown C. 
fascians and bacterial isolate, FBR-72-65, "" 



C. fascians C. fascians FBR-72-65 
Test Berqey's Manual "~ICPP CF-19 



Gelatin lignification 

Nitrate reduction + « 

Starch hydroylis 

Hydrogen sulfide + + 



Litmus milk blued + 



+. 
+ + 



Acid formation 
Dextrose 
Maltose 

Xylose + 

Lactose 



+ + + 

+ + + 



Gram stain + 



+ 



+ + 



Optimum temperature 25-28 C 25-30 C 25-30 C 



DISCUSSION 

Tobacco plants may be severely stunted and losses 
in yield may result if the tobacco plants become infected 
with FBR soon after transplanting. The size plant used 
for transplanting is easily inoculated with the causal 
agent of FBR. If the seedbed is contaminated, this may 
be the source of inoculum for previously uninfested 
fields. In greenhouse experiments FBR was easily trans- 
mitted when tobacco plants were planted in soil from a 
FBR infested field. Old, dried, fasciated tissue is a 
good source of FBR inoculum. 

No differences were found between any of the sources 
of FBR. When they were inoculated on tobacco plants in 
a greenhouse, the symptoms expressed were identical. All 
would form small tobacco plants from fasciated tissue 
near the soil surface. Thompson (19) did not find this 
to be true in earlier experiments. 

Tobacco plants could be inoculated with macerated 
tissue from any type of fasciated tissue and FBR was 
transmitted. This inoculum could be filtered and diluted 
i:iO and still be viable. All bacteria could be removed 
from this filtrate, and FBR symptoms could still be repro- 
duced on tobacco plants. 

This disease h^.3 been reported in Florida (24), 

40 



41 

North Carolina (8), Georgia (6), Maryland (l«), and 
Central Ar.arica (i:,i, lb;. Many researchers have tried 
and failed to determine the causal agent (3, 8, 17). 
It was proposed that FBR is caused by the bacterium C. 
fascians (8, 12, 17, 19, 22;. Worlcing on this premise, 
organisms other than a bacterium were ruled out, and 
isolation techniques for the isolation of a bacterium 
were employed using a medium similar to one suggested 
by Burkholder (2). The medium was the same except 
Difco PDA and 300 mg potassium dichromate were used. On 
this medium C. fascians is allowed to grow without being 
overgrown by faster growing saprophytic organisms. 
After several attempts at isolation a bacterium was 
isolated that reproduced FBR symptoms when inoculated 
on tobacco. This same bacterivim was reisolated. 

Several physiological tests were conducted on the 
isolate, FBR-72-65, from fasciated tobacco tissue as well 
as a C. fascians isolate from M. P. Starr. They all 
agreed with the results in Bergey's Manual except the 
nitrate reduction test. Starr's C. fascians isolate did 
not agree with Sergey's Manual in this respect. Based 
on these tests and growth characteristics the isolate 
was identified as C. fascians. 



SUMMARY 

FBR is a very easily transmitted disease and no 
extra or excessive injury to the root systems of 
tobacco plants is necessary. Ample injury is inflicted 
to a tobacco plant during normal transplanting procedures. 

Any type of gall tissue can be used to transmit 
the causal agent of FBR. Dried tissue is also effective. 
This means that FBR can easily be carried over from one 
growing season to another. 

The temperature optimum for plant growth, 25-30 C, 
is optimum for FBR development, and almost any size or 
age of plant can be infected. At least 4 weeks are 
required for expression of symptoms. 

All Nicotiana species tested were susceptible to 
FBR as well as some 14 cultivars of N. tabacum. Cauli- 
flower was found to be naturally infected with FBR in 
Central America. This FBR was easily transmitted to 
tobacco and back to cauliflower. Sweet potato, cabbage, 
and sweet pea were also suscepts to the disease. 

Increasing fertilization levels did not cause FBR 
to be initiated sooner but did result in a faster develop- 
ment of the fasciated tissue. This could explain why 
FBR is more severe on burley tobacco than flue-cured 
since burley received extremely high rates of fertilizer. 

42 



43 

A bacterium was isolated from fasciated tissue 
from Central America, and this bacterium produced FBR 
symptoms on tobacco plants in the greenhouse. The 
same bacterium was reisolated from these plants using 
a medium containing 40 g Difco PDA, 2 g Na2HP04, 2 g NaCl, 
1 g sodium citrate, 1 g asparagine, and 300 mg potassium 
dichromate per liter of water. This bacterium was 
identified as Corynebacterium fascians. Two colony 
types were present, a rough colony which was not 
pathogenic and a smooth type which was pathogenic. 

More work is need with this disease and the causal 
agent. Since the causal agent was found to be a bacterixim, 
a control method can probably be developed. In as much 
as the disease is not at present a wide spread economic 
problem, the control would probably be with chemicals 
and/or crop rotation. The possibility of a wild host 
should also be investigated as well as cultural and 
environmental conditions that may favor the disease and 
influence its severity. 



LITERATURE CITED 



1. Baker, Kenneth F. 1950. Bacterial fasciation 

disease of ornamental plants in California. 
Plant Dis. Rsptr. 34i 121-125. 

2. Burkholder, Walter I-I. 1938. The occurrence in 

the United Szates of the tuber ring rot and 
wilt of the potato. Aarier. Potato J. 15:243-245. 

3. Dukes, P. D., S. ?. Jenkins, Jr., and R. W. Toler. 

1963. La improved inoculation technique for 
transmission of false broomrape to flue-cured 
tobacco. Plant Dis. Reptr. 47:895-897. 

4. Dukes, P. D,, S. F. Jenkins, Jr., and R. W. Toler. 

1964. Influences of soil temperature on the 
development of false brocvirape on the roots of 
flue-cured tobacco. Phytopathology 54:622. 
(Abstr . ) 

5. Jacobs, S. E. and U. Mohanty. 1951. Studies in 

bacteriosis XXVII. Factors influencing infection 
'^y C o " y n a ba c t e r i ur a fas cians (Tilford) Dowson. 
•Ann. Appi. Biol. S'SsJjS. 

6. Jenkins, S« F., Jr., P. D. Dukes, and S. S. Thompson. 

1963. Flue-cured tobacco diseases in Georgia, 
1959-62. Plant Dis. Reptr. 47:9:^98. 

7. Johnson, J. T. and L. W. Nielsen. 1970. Sweet 

potato a suscepa of false broomrape. Plant 
Dis. Reptr. 54; 97^-980. 

8. Kel.-.an, A., G. B. Lucas, and K. R, Garriss. 1955. 

F.-lse broor.-irapa of tobacco in North Carolina. 
E:.ij:.^ Mitchell Sci. See. J. 71:173-174. (Abstr.) 

9. Kado, C. I. and M. G. Heskett. 1970. Selective 

media for _3olation of Agrobacterium , 
Corynebacteriu.-.. , Erwinia , Pseudomonas , and 
Xanthoracnas . Phytopathology 60:969-976. 



44 



45 



10. Lacey, M. S. 1936. Studies in bacteriosis XXII. 

I. The isolation of a bacterium associated with 
the fasciation of sweet peas, "cauliflower" 
strawberry plants and leafy gall of various 
plants. Ann. Appl. Biol. 23:302-310. 

11. Lacey, M. S. 1939. Studies in bacteriosis XXIV. 

Studies on a bacterium associated with leafy 
galls, fasciations and "cauliflower" disease 
of various plants. Part III. Further inoculation 
experiments and cultural studies. Ann. Appl. 
Biol. 26«262-278. 

12. Lucas, G. B. 1958. Diseases of tobacco. 

Scarecrow Press, New York, 498 p. 

13. Massey, Ira. 1970. (Personal communication) 

14. Marcelli, E. 1950. Carattere endemico dei 

"tumori radicali" presumibilimente da. Bact. 
tumefasciens, in campo di tabacco. (Nota 
preliminare) Tabacco 54i 199-201. 

15. Miller, C. R, and G. B. Lucas. 1970. False 

broomrape, a destructive tobacco disease in 
Nicaragua. Phytopathology 60t 1541 . (Abstr.) 

16. Mohanty, U. 1951. Coryne bacterium fascians 

(Tilford) Dowsonj Its morphology, physiology, 
nutrition and taxonomic position. Trans. Brit. 
Mycol. Soc. 34» 23-24. 

17. Newman, M. A. and R. W. Toler. 1970. Characteristics 

of false broomrape disease and properties of the 
etiological agent. Phytopathology 50t 1305-13U6. 
(Abstr. ) 

18. SJcoog, H. A. 1963. Diseases of Maryland tobacco. 

Maryland Agr. Exp. Sta. Bull. 419. 12 p. 

19. Thompson, Samuel Stanley, Jr. 1965. False broomrape 

of tobacco: anatomy and morphology of tumors) 
influence of environment; reaction of some 
Nicotiana species to the disease. Master's 
Thesis, Purdue University. 81 p. 

20. Tilford, P. E. 1936. Fasciation of sweet pea 

caused by Phytomonas fascians n. sp. J. Agr. Res. 
53:383-4107"^ 

21. Trotter, A. 1946, Sulla presenza di tumori radicali 

nelle coltivanzioni di tabacco di pieno campo. 
Ric. Ossvz. Divulg. fitopat. Campania ed 
Mezzogiorno (Portici) 10:65-80. (Abstr. Rev. 
App. Mycol. 26:34. 1947) 



46 



22. Vallenu, W. D. 1953. False broomrape and leaf 

curl I Two new tobacco diseases in Kentucky, 
Plant Dis. Reptr. 37i 538-539. 

23. Valleau, W. D,, E, M. Johnson, and S. Diachun. 

1954. Tobacco diseases. Kentucky Agr. Exp. Sta. 
Cir. 522. 55 p. 

24. Weber, G. F. 1954. False broomrape of tobacco in 

Florida. Plant Dis. Reptr. 38»121. 



BIOGRAPHICAL SKETCH 

Wayne Clark Mixson was born on a farm in Marion 
County, Florida, on November 29, 1944. He completed 
twelve years of school at Reddick, Florida, and was 
graduated from North Marion High School in 1962. He 
attended the University of Florida at Gainesville. In 
1965 he received the degree of Bachelor of Science in 
Agriculture with a major in agronomy and in 1969 the 
degree of Master of Science in Agriculture with a major 
in agronomy. In September, 1969, he began studies 
toward the degree of Doctor of Philosophy. He is a 
member of the American Phytopathological Society, the 
American Society of Agronomy, Crop Science Society of 
America, and the Florida Turf-Grass Association. He 
is employed as a research agronomist by 0. M. Scott and 
Sons, Apopka, Florida. 

Wayne C. Mixson is married to the former Sharon 
Kay Freimuth of Lowell, Florida, a librarian with a 
Master of Science degree in Library Science. 



47 



t"HM 



I cortify thc^t I have read this study and that in 
my opinion it conforrus to accepta'oie standards of 
scholarly presentation and is fully adequate, in scope 
and quality, as a dissertation for the degree of Doctor 
of Philosophy. 




Joert E. 
Chairman 
Professor o 
Pathology 



Stall, 



Plant 



I certify -chat I have read this study and that in 
L\y opinion it conforms to acceptable standards of 
scholarly presentation and is fully adequate, in scope 
and quality, as a dissertation for the degree of Doctor 
of Philosophy. 



\Aa^<A 




Laurence H. Puray 
Professor & Chairman 
of Plant Pathology 




I certify that I have read this study and that in 
my opinion it conforms to acceptable standards of 
scholarly presentation and is fully adequate, in scope 
and quality, as a dissertation for the degree of Doctor 
of Philosophy. 



jA.^t^^^/^y 



7ernon G. ' Perry 
Professor of Entomology 

and Nematology 




I cortify thi.t I have read this study z-.-id that in 
r.y opinion it cc.aforns u-^ acceptable standards of 
scholarly presentation and is fully adequate, in scope 
and qur.lity, as a dissertation for tha dc^-ree of Doctor 
of Philosophy. 



-■"rca Clark \ 
Professor of 
AgronoiTiy 



This dicwv-rtation vas subr;.itted to the Dean of the College 
of Agriculture and to the Graduate Council, and was 
accepted as partial fulfill^r.ent of the requirements for 
t..3 degree of Doctor of Philosophy, 

DeceiuL'Dr, 1^72 « 




.gri.cui.wur 



Daan, Graduate School